Effect of pH and protein composition on proteolysis of goat milk proteins by pepsin and pancreatin.

The roles of protein composition, pH and enzymes in goat milk protein hydrolysis is still unclear and the proteolysis of low abundant goat milk proteins has received limited attention. The aim of this study was to study the impact of protein composition and proteolytic conditions on goat milk protein hydrolysis in a simplified digestion model. Both whole milk and infant formula were hydrolyzed at pH 2 and 4, using pepsin as well as pepsin combined with pancreatin. Intact proteins were separated from digests using spin filters, followed by bottom-up proteomics of the separated proteins. Results show that under all conditions, caseins are hydrolyzed quickly. Goat casein hydrolysis in infant formula was slightly faster than in goat whole milk, possibly due to less casein coagulation during pepsin hydrolysis at both pH 2 and 4. Several low abundant immunoactive goat milk proteins, especially immunoglobulins, GLYCAM-1 and osteopontin, resisted proteolysis more than high abundant proteins, independent of the pH and enzyme used for hydrolysis. Fast hydrolysis of casein and slow hydrolysis of immunoactive proteins may indicate a good balance between protein utilization and protection of the infant by goat milk proteins.

The role of orally ingested milk fat globule membrane on intestinal barrier functions evaluated with a suckling rat pup supplementation model and a human enterocyte model.

Milk fat globule membrane (MFGM), the membrane surrounding secreted fat droplets in milk, contains components involved in a wide range of bioprocesses including cell proliferation and differentiation. The intestine is relatively immature and permeable at birth. Since MFGM is partly resistant to digestion in infancy, we hypothesized that orally ingested MFGM promotes intestinal development by enhancing intestinal barrier functions in early life. An established suckling rat model was used; Sprague-Dawley rats were bred, and litters were culled to 10 pups/dam. Pups were supplemented orally with MFGM (0, 100, or 300 mg/kg/d) from postnatal day 1-20. Intestine samples were collected for histology, real-time quantitative PCR, immunoblotting, and immunohistochemistry analysis. Additionally, differentiated Caco-2 cells were used to assess effects of MFGM on the human intestinal barrier. Control and MFGM-supplemented rat pups showed similar growth. Intestinal differentiation and expression of tight junction proteins in jejunum and colon were significantly increased by orally ingested MFGM, and MFGM supplementation significantly activated PI3K/Akt/mTOR, mitogen-activated protein kinases, and myosin light chain kinase signaling pathways, suggesting that MFGM promotes intestinal development by triggering various signaling pathways. In human enterocytes (polarized Caco-2 cells), MFGM (400 µg/mL for 72 h) decreased permeability, as revealed by increased transepithelial electrical resistance. In Caco-2 cells, MFGM also enhanced expression of tight junction proteins, including claudin-4 and ZO-2. In conclusion, orally ingested MFGM may exert beneficial roles in intestinal development by activating various cell signaling pathways to upregulate tight junction proteins and thereby increasing intestinal barrier functions.

Biological activities of commercial bovine lactoferrin sources.

Lactoferrin (Lf) samples from several manufacturers were evaluated in vitro. The purity and protein form of each Lf were examined by SDS-PAGE, Western blot, and proteomics analysis. Assays were conducted to evaluate uptake of Lfs and iron from Lfs by enterocytes as well as Lf bioactivities, including effects on intestinal cell proliferation and differentiation, IL-18 secretion, TGF-ß1 transcription, and growth of enteropathogenic Escherichia coli (EPEC). Composition of the Lfs varies; some only contain a major Lf band (∼80 kDa), and some also contain minor forms. All Lfs and iron from the Lfs were absorbed by Caco-2 cells, with various efficiencies. The bioactivities of the Lfs varied considerably, but there was no consistent trend. All Lfs promoted intestinal cell proliferation, secretion of IL-18, and transcription of TGF-ß1. Some Lfs exhibited pro-differentiation effects on Caco-2 cells. Effects of pasteurization (62.5 °C for 30 min, 72 °C for 15 s, or 121 °C for 5 min) on integrity, uptake, and bioactivities were examined using Dicofarm, Tatua, and native bovine Lfs. Results show that pasteurization did not affect protein integrity, but variously affected uptake of Lf and its effects on intestinal proliferation, differentiation, and EPEC growth. To choose a Lf source for a clinical trial, assessment of bioactivities is recommended.

Evaluation of Bioactivities of the Bovine Milk Lactoferrin-Osteopontin Complex in Infant Formulas.

Human milk contains several bioactive proteins, including lactoferrin (LF) and osteopontin (OPN). These two proteins have been shown to form a complex, which shows increased bioactivities. Bovine LF and OPN can also form such a complex. We assessed bioactivities of the bovine LF-OPN complex (at molar ratios of LF:OPN = 3:1, 5:1, or 8:1) in a formula protein matrix, including LF, OPN, bovine whey protein hydrolysate, and α-lactalbumin. Our results show that the bovine LF-OPN complex together with formula proteins is resistant to in vitro digestion, stimulates intestinal cell proliferation (by 15-50%) and differentiation (by 30-50%), increases antibacterial activity (by 25-50%), and enhances intestinal immunity. The 3:1 ratio of LF to OPN exhibits the most potent effects, as compared with the other two ratios. In conclusion, adding bovine LF and OPN to infant formulas may result in increased stability of the two components and enhanced bioactivities, possibly improving outcomes in formula-fed infants.

Exosomal MicroRNAs in Milk from Mothers Delivering Preterm Infants Survive in Vitro Digestion and Are Taken Up by Human Intestinal Cells.

SCOPE: This study investigates the ability of preterm milk exosomes to survive gastric/pancreatic digestion, internalization by intestinal epithelia, and the microRNAs (miRNAs) contents. METHODS AND RESULTS: At average infant age 1 week and 6 days, milk is collected from mothers who delivered preterm and term infants (n = 10). Milk is exposed to conditions simulating infant gut digestion. Exosomes are isolated and lysed, and the exposed miRNAs are sequenced. Preterm milk exosomes survive in vitro digestion, and can be taken up by intestinal epithelia. Three hundred and thirty miRNAs are identified as preterm milk exosome miRNAs, and in vitro digestion does not have a pronounced effect on their expression. The abundant miRNAs in preterm milk exosomes are similar to those from term milk. Twenty-one low abundance miRNAs are specifically expressed in preterm milk exosomes compared to early term milk in the current study and what previously is found in mature term milk. CONCLUSION: These results for the first time reveal the survivability of preterm milk exosomes following simulated gastric/pancreatic digestion. The authors demonstrate the richness of the miRNAs content in these exosomes. The results improve the knowledge of preterm milk biology and the molecular basis by which exosome miRNAs may uniquely affect preterm infants during early development.

Human milk exosomes and their microRNAs survive digestion in vitro and are taken up by human intestinal cells.

SCOPE: Human milk exosomes provide a natural means of genetic material transfer to infants; however, the effect of gastric/pancreatic digestion milk exosomes stability and their microRNA content is largely unknown. METHODS AND RESULTS: We took a simulated gastric/pancreatic digestion protocol to perform in vitro digestion of milk exosomes, explore intestinal epithelial uptake, and further elucidate microRNA responses to digestion at early-, mid-, late lactation by massive parallel sequencing. Both undigested and digested exosomes enter human intestinal crypt-like cells (HIEC), with evidence of nuclear localization. We identified 288 mature microRNAs from all 24 exosome samples, and an additional 610 at low abundance. A large number of synapse development- and immune-related microRNAs were identified. hsa-miR-22-3p was the most abundant microRNA, and the top 15 microRNAs contributed ∼11% of the sequencing reads. Upon digestion, the overall microRNA abundance in human milk exosomes was stable. CONCLUSION: Our results for the first time reveal the survivability and complexity of human milk exosome microRNAs upon simulated gastric/pancreatic digestion, and the dynamics during lactation stages. The results suggest a previously underexplored area of infant response to genetic material in human milk exosomes.

Lactoferrin and the lactoferrin-sophorolipids-assembly can be internalized by dermal fibroblasts and regulate gene expression.

Lactoferrin (Lf) is an iron-binding multifunctional protein, mainly present in external secretions. Lf is known to penetrate skin and may thus exert its multiple functions in skin. Sophorolipids (SLs) are glycolipid biosurfactants, which have been shown to enhance absorption of commercial bovine Lf (CbLf) in model skin via forming an assembly with CbLf. In this study, uptake and post-internalization localization of bovine Lf (bLf), CbLf, and human Lf (hLf) with or without forming assemblies with SLs in human dermal fibroblasts (HDFn) were determined using 125I-labeled Lfs and confocal microscopy, respectively. Our results show that all 3 Lfs were internalized by HDFn; although SLs did not significantly affect the uptake of Lfs, it changed Lf localization by accumulating Lfs in the perinuclear region. Furthermore, microarrays were used to investigate transcriptional profiling in HDFn in response to CbLf, SLs, or CbLf-SLs-assembly treatments. Transcriptome profiling indicates that CbLf may play roles in the protection of skin from oxidative stress, immunomodulatory activities, and enhancement of wound healing. The assembly had similar effects but dramatically modulated the transcription of some genes. SLs alone modified signaling pathways related to lipid metabolism, as well as synthesis of sex hormones and vitamins. Thus, CbLf may exert beneficial effects on skin, and these effects may be modulated by SLs.

Comparison of bioactivities of talactoferrin and lactoferrins from human and bovine milk.

OBJECTIVES: Lactoferrin (Lf) is an iron-binding glycoprotein present in high concentration in human milk. It is a pleiotropic protein and is involved in diverse bioactivities, such as stimulation of cell proliferation and differentiation, immune competence, antimicrobial activities, anti-infection, and anticancer activities. Lf has been shown to be partly resistant to proteolysis in the gastrointestinal tract and may thus play important roles in the intestine and liver during infancy. Talactoferrin alfa (TLf) is a recombinant human Lf shown to protect against sepsis and necrotizing enterocolitis as well as cancer. Because bovine Lf (bLf) and human Lf have different amino acid composition and all 3 Lfs differ in glycosylation, they may have different functions/potency. The objective of the present study was to investigate and compare bioactivities of TLf and Lfs from human and bovine milk and thus to provide a better understanding of the bioactivities of different forms of Lf and their potential applications. METHODS: In the present study, Caco-2 and C3A cells were used as intestine and liver models to evaluate internalization of Lfs by intestine and liver cells, effects of Lfs on cell proliferation and differentiation, growth of enteropathogenic Escherichia coli (EPEC), chemokine (C-C motif) ligand 20 (CCL20) secretion, and transforming growth factor (TGF)-ß1 expression. In addition, HT-29 cells were used as a colon cancer cell model to examine the effects of Lfs on apoptosis. RESULTS: All Lfs significantly enhanced cell proliferation and differentiation, apoptosis, CCL20 secretion, and TGF-ß1 expression. They also markedly suppressed growth of EPEC. Compared with bLf, TLf showed stronger effects on suppression of EPEC growth and enhancement of TGF-ß1 secretion, whereas bLf exhibited more potent effects on cell differentiation, apoptosis, and CCL20 secretion. CONCLUSIONS: Our results demonstrate that TLf has several bioactivities similar to human Lf and bLf from milk and may play critical roles in immune and intestinal development in infants as well as having anti-cancer activities in adults. TLf and bLf may be used for different applications owing to their various potencies. TLf may preferentially be used for anti-bacterial applications, whereas bLf may be used for cancer therapy because it exhibits stronger effects on CCL20 secretion, cell differentiation, and apoptosis.

Effect of phytate reduction of sorghum, through genetic modification, on iron and zinc availability as assessed by an in vitro dialysability bioaccessibility assay, Caco-2 cell uptake assay, and suckling rat pup absorption model.

Improved iron and zinc availability from sorghum, a commonly consumed staple, will benefit many malnourished communities in rural Africa burdened with high prevalence of iron and zinc deficiency. This research compared the effect of genetic phytate reduction in sorghum on iron and zinc bioaccessibility and uptake measured by in vitro dialysability and Caco-2 cell uptake assays to that of iron and zinc absorption measured by a suckling rat pup model. The phytate reduction (80-86%) in these sorghums significantly increased zinc availability. The Caco-2 cell method, but not the dialysability assay, proved useful in estimating zinc absorption. The measured increase in iron availability differed between the methods, possibly due to the effect of varying mineral (Ca, Fe, Zn, P) contents of the sorghums. This effect was most prominent in the iron uptake results. More research is needed to determine the effect of naturally occurring variations in mineral contents of sorghum on the iron uptake by Caco-2 cells.

Biofortification of rice with zinc: assessment of the relative bioavailability of zinc in a Caco-2 cell model and suckling rat pups.

Staple foods, such as rice, can now be enriched in micronutrients through conventional breeding (i.e., biofortification) to enhance dietary intake of vulnerable populations. The objectives of this study were (1) to establish a rapid, high capacity Caco-2 cell model to determine the relative bioavailability of zinc (Zn) from samples of staple food breeding lines for potential use as a guideline for selection/breeding and (2) to determine the relative bioavailability of Zn from conventional rice varieties and one Zn-biofortified type. Polished or undermilled, parboiled rice samples were digested in vitro with pepsin and pH adjustment, and by pancreatic enzymes. Zn uptake from digested samples was measured in Caco-2 cells in culture. A previously validated rat pup model was also used to assess Zn absorption in vivo, using gastric intubation and (65)Zn labeling. Pups were killed after 6 h, and radioactivity in tissues and in small intestine perfusate and cecum-colon contents was used to measure Zn bioavailability. A biofortified rice variety contained substantially more Zn than conventional varieties, with no change in phytate content. Absorbed Zn (µg/g rice) was significantly higher from the new variety in both the in vitro Caco-2 cell model (2.1-fold) and the rat pup model (2.0-fold). Results from the two models were highly correlated, particularly for the polished samples. Biofortification of rice with Zn results in significantly increased Zn uptake in both models. Since results from the Caco-2 cell model correlated well with those from rat pups, this cell model is likely to predict results in human populations and can be used for screening purposes.

miR-214 regulates lactoferrin expression and pro-apoptotic function in mammary epithelial cells.

Lactoferrin (Lf) is an abundantly expressed protein in human milk. Lactoferrin exhibits several important biological functions, and its expression is regulated by multiple environmental factors. Cellular endogenous factors, however, have not been extensively studied with regard to lactoferrin gene expression. In this study, we showed that lactoferrin gene expression and function are directly targeted by miR-214 in HC11 and MCF7 cells. In the lactoferrin mRNA 3 prime untranslated region (UTR) of human, mouse, rat, pig, bovine, camel, and goat species, there is a conserved region that perfectly matches the seed region of miR-214. Transfection of miR-214 mimic in HEK293 cells dose-dependently inhibited the activity of pGL3-control vector containing lactoferrin mRNA 3 prime UTR downstream of the luciferase gene. In HC11 cells, miR-214 overexpression inhibited the induction of lactoferrin expression by beta -estradiol (E2) and dexamethasone-prolactin-insulin (DPI). Furthermore, in MCF7 cells, overexpression of miR-214 markedly decreased lactoferrin expression (P lt 0.05), and inhibition of endogenous miR-214 expression increased lactoferrin expression and cellular apoptotic activities (P lt 0.05). In summary, our data showed that miR-214 is directly involved in lactoferrin expression and lactoferrin mediated cancer susceptibility (proapoptotic activities) in mammary epithelial cells.

Amelioration of doxorubicin-induced cardiac and renal toxicity by pirfenidone in rats.

PURPOSE: Doxorubicin (DXR) is an anthracycline glycoside with a broad spectrum of therapeutic activity against various tumors. However, the clinical use of DXR has been limited by its undesirable systemic toxicity, especially in the heart and kidney. This study was designed to test the effectiveness of dietary intake of pirfenidone (PD) against DXR-induced cardiac and renal toxicity. METHODS: Male Sprague Dawley rats were placed into four treatment groups: saline injected intraperitoneally (i.p.) plus regular diet (SA+RD); DXR i.p. plus regular diet (DXR+RD); saline i.p. plus the same diet mixed with 0.6% PD (SA+PD); and DXR i.p. plus the same diet mixed with 0.6% PD (DXR+PD). The animals were fed regular or regular plus PD diets 3 days prior to i.p. injections of either saline or DXR and continuing throughout the study. A total dose of DXR (16.25 mg/kg) or an equivalent volume of saline was administered in seven injections (2.32 mg/kg per injection) three times per week with an additional dose on the 12th day. At 25 days following the last DXR or saline injection, some animals were anesthetized for the measurement of cardiac and pulmonary function, and others were killed by an overdose of pentobarbital. At the time the animals were killed, abdominal fluid was collected. Kidney and heart were removed, weighed, fixed with 10% formalin or frozen in liquid nitrogen. The fixed tissues were used for histological examination and the frozen tissues were used for biochemical studies. RESULTS: The average volumes of abdominal fluid in the DXR+RD and DXR+PD groups were 9.42 ml and 3.42 ml and the protein contents of abdominal fluid in the DXR+RD and DXR+PD groups were 218 mg and 70 mg, respectively. A 12.5% mortality occurred in the DXR+RD group as compared to 0% in DXR+PD group. There were no changes in any of the cardiac or pulmonary physiological parameters in any of the four groups. The changes in the heart and kidney of the DXR+RD group included reduction in organ weight, increase in hydroxyproline content of heart, increase in hydroxyproline, and lipid peroxidation in the kidney and plasma, and increase in protein concentration in urine as compared to rats in the control, SA+RD and SA+PD groups. Treatment with PD abrogated the DXR-induced increases in hydroxyproline content in the heart and kidney, lipid peroxidation of the kidney and plasma, and protein content of the urine in the DXR+PD group. DXR treatment alone caused disorganization of cardiac myofibrils, vacuolization of the myofibers, and renal tubular dilation with protein casts in both the cortical and medullary regions. Treatment with PD minimized the DXR-induced histopathological changes of heart and kidney in the DXR+PD group. CONCLUSIONS: Treatment with PD reduced the severity of DXR-induced toxicity as assessed by reduced mortality, diminished volume of recovered fluid in the abdominal cavity, and severity of cardiac and renal lesions at both the biochemical and morphological levels. These results indicate that PD has the potential to prevent DXR-induced cardiac and renal damage in humans on DXR therapy.